The importance of the factor VIIIa-factor IXa enzyme complex is illustrated by hemophilia in which the absence of either protein causes life-threatening bleeding. In our investigations over the past 5 yrs we have found that on membranes with low mole fractions of acidic phospholipid, resembling cell membranes, factor VIII exhibits stereoselective affinity for phosphatidyl-L-serine containing binding sites. The phosphatidylserine (PS)-containing sites are highly specific so that no plasma proteins will compete efficiently with factor VIII for binding. We have also found that factor VIIIa binds to factor IXa with moderately high affinity in the absence of phospholipid membranes but the catalytic efficiency of the factor VIIIa-factor IXa complex is greatly enhanced by binding to PS containing membranes. These findings lead us to the following three hypotheses. First, membrane-bound factor VIIIa provides high affinity membrane binding sites for factor IXa and high affinity binding requires the correct conformation of the factor IXa active site. We will measure binding of factor IXa, derivatized in the active site by a fluorescein-labeled tripeptide substrate (Glu-Gly- Arg-chloromethyl ketone), to factor VIIIa that is bound to phosphatidyl- L-serine-containing synthetic membranes vs. platelets. For comparison factor IXa will be derivatized with a non-physiologic tripeptide substrate (D-Phe-Pro-Arg chloromethyl ketone) that alters the active site conformation of thrombin and apparently of factor IXa. Second, individual phospholipid molecules or small aggregates of phospholipids activate the factor VIIIa-factor IXa complex by causing a change in the conformation of the enzyme complex. We propose experiments with soluble phospholipids and with phospholipids in mixed micelles to distinguish moieties that activate the factor VIIIa-factor IXa complex from those that are inert constituents of the membrane matrix. Experiments with antibodies to the phospholipid-binding structures of factor IXa and factor VIIIa, and with modified forms of factors X, will indicate whether the complex is altered at a site distant from the membrane. Third, the high specificity of PS-containing membranes for factors VIIIa results from spontaneous arrangement of membrane phospholipids into microdomains which function as specific binding sites on platelet membranes. We will prepare monoclonal antibody fragments that recognize PS-containing microdomains in order to better characterize microdomains and to investigate their importance as functional receptors for factors VIII. Completion of the proposed studies will substantially increase our understanding of the role of the platelet phospholipids in activating the factor VIIIa-factor IXa complex.